Miniaturized Modular Click Chemistry-enabled Rapid Discovery of Unique SARS-CoV-2 M^pro Inhibitors With Robust Potency and Drug-like Profile

The COVID-19 pandemic has required an expeditious advancement of innovative antiviral drugs. In this study, focused compound libraries are synthesized in 96- well plates utilizing modular click chemistry to rapidly discover potent inhibitors targeting the main protease (M^pro) of SARS-CoV-2. Subsequent direct biological screening identifies novel 1,2,3-triazole derivatives as robust M^pro inhibitors with high anti-SARS-CoV-2 activity. Notably, C5N17B demonstrates sub-micromolar M^pro inhibitory potency (IC₅₀ = 0.12 µM) and excellent antiviral activity in Calu-3 cells determined in an immunofluorescence-based antiviral assay (EC₅₀ = 0.078 µM, no cytotoxicity: CC₅₀ > 100 µM). C5N17B shows superior potency to nirmatrelvir (EC₅₀ = 1.95 µM) and similar efficacy to ensitrelvir (EC₅₀ = 0.11 µM). Importantly, this compound displays high antiviral activities against several SARS-CoV-2 variants (Gamma, Delta, and Omicron, EC₅₀ = 0.13 - 0.26 µM) and HCoV-OC43, indicating its broad-spectrum antiviral activity. It is worthy that C5N17B retains antiviral activity against nirmatrelvir-resistant strains with T21I/E166V and L50F/E166V mutations in M^pro (EC₅₀ = 0.26 and 0.15 µM, respectively). Furthermore, C5N17B displays favorable pharmacokinetic properties. Crystallography studies reveal a unique, non-covalent multi-site binding mode. In conclusion, these findings substantiate the potential of C5N17B as an up-and-coming drug candidate targeting SARS-CoV-2 M^pro for clinical therapy.